Generally, mass spectrometers measure mass-to-charge ratios of charged samples, enabling contents of the samples to be identified. Use of mass spectrometers has been expanded to include identification of proteins and corresponding peptides. This requires ions of a protein in the sample to be volatilized, in accordance with a variety of volatilizing techniques, such as electrospray ionization (ESI) and matrix-assisted laser desorption and ionization (MALDI) and provided to a mass analyzer of the mass spectrometer. The proteins and peptides may then be identified, for example, by matching the measured mass-to-charge ratios to a database of mass-to-charge rations of known proteins and peptides. Tandem mass spectrometry (MS/MS) provides multiple stage measurements of a sample, for example, using separate analyzers corresponding to the multiple stages, or using a single analyzer to analyze the sample multiple times.
Currently, powerful computer processing and enhanced performance of bioinformatics tools that analyze mass spectrometry data make it possible to match results of an MS/MS scan of a sample to a peptide in real-time. That is, the peptide may be identified in a timescale comparable to the time between two successive acquisition events of a mass spectrometer (i.e., the time it takes to acquire one spectrum).
Bottom-up acquisition protocols for MS/MS have increased sample coverage by applying different rules on how to select the most intense precursor ions of a sample for further MS/MS acquisition. For example, rules on ion intensities have been used whereby a precursor ion exclusion list may be built based on information collected on the first of two consecutive runs of a sample, known as repetitive liquid chromatography (LC)-MS/MS. While repetitive LC-MS/MS extends coverage, it also doubles acquisition time, thus becoming impractical in high throughput workflows.